Search

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K+- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca2+ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca2+ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca2+ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca2+ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca2+ sensitization. These results suggest that paracetamol might be a new bronchodilator. Keywords: Paracetamol, Airway smooth muscle, Canonical transient receptor potential channel, L-type voltage dependent Ca2+ channel, Store-operated Ca2+ channel, Ca2+ sensitization

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

S100 calcium binding protein A9 (S100A9) is involved in a variety of biological processes such as inflammation and tumor cell migration and invasion regulation. The purpose of this study was to construct S100A9 gene-edited mice by using CRISPR/Cas9 technology, thereby providing an animal model for exploring the biological functions of this gene. According to the S100A9 gene sequence, the single-stranded small guide RNA (sgRNA) targeting exons 2 and 3 was transcribed in vitro, and a mixture of Cas9 mRNA and candidate sgRNA was injected into mouse fertilized eggs by microinjection. Early embryos were obtained and transferred to surrogate mice, and F

Characterized by abnormal smooth muscle contractility and airway inflammation, asthma is one of the most common airway diseases worldwide. Diacerein is a well-known anti-inflammatory drug, widely used in osteoarthritis. In current study, the innovative usage of diacerein in anti-contractile and anti-inflammatory treatment of asthma was studied. In vitro experiments including tension measurement and patch-clamp technique and in vivo experiments including establishment of mice model and measurement of respiratory resistance were applied to explore the role of diacerein in asthma. It turned out that agonist-precontracted mouse airway smooth muscle could be relaxed by diacerein via intracellular and extracellular calcium mobilization which was mediated by switched voltage-dependent L-type Ca2+ channels, non-selective cation channels, large-conductance Ca2+-activated K+ channel, and Na+/Ca2+ exchangers. Furthermore, diacerein could relieve bronchospasm and control airway inflammation in asthmatic mice via reduction of several inflammatory factors. Our studies elucidated the potential therapeutic property of diacerein in asthma treatment and the possible underlying mechanism. It also confirmed that new uses for already-approved drugs could be an important form of innovation.

Cancer cell resistance to current anticancer therapeutics as well as the side effects are still obstacles to successful cancer therapy. Hence, the development of novel anticancer agents or therapeutics is of vital significance, and especially rational adjuvant therapies containing low-cost natural products with multiple targets have attracted great interests. Triptolide, the main biocomponent of Tripterygium wilfordii Hook F, is restricted in clinical applications mainly due to its severe systemic toxicities, although it has shown strong antitumor activities in preclinical studies. Mounting evidence suggests that triptolide at low doses as an adjuvant therapeutic agent circumvents resistance to current anticancer therapies, enhances the anticancer effectiveness, and relieves toxicities of both triptolide and anticancer therapies. Furthermore, several unique antitumor targets of triptolide make it superior to other therapeutics. The molecular mechanisms of triptolide-induced anti-resistance and sensitization effects include changes in ATP-binding cassette transporters, induction of apoptosis pathways, increase in tumor suppressors and decrease in oncogenic factors, and interactions with the RNA polymerase II complex; targeting cancer stem cells and tumor-microenvironment-mediated resistance are also involved. Besides, some synthetic derivatives and novel delivery systems of triptolide are also developed to enhance the water-solubility and reduce the toxicity, which will also be discussed.

This study aimed to elucidate the mechanisms of nuciferine (a main aporphine alkaloid of lotus leaf extract), which can induce relaxation in contracted tracheal rings. Under Ca2+-free and 2 mM Ca2+ conditions, we found that nuciferine had no effect on the resting muscle tone of tracheal rings. In contrast, nuciferine relaxed high K+-contracted mouse tracheal rings in a dose-dependent manner and inhibited both Ca2+ influx and voltage-dependent L-type Ca2+ channel currents induced by high K+. Similarly, nuciferine also inhibited acetylcholine-induced contractions in mouse tracheal rings in a dose-dependent manner. Meanwhile, both acetylcholine-induced intracellular Ca2+ influx and whole-cell currents of nonselective cation channels were blocked by nuciferine. Together, the results indicate that nuciferine-induced relaxation in tracheal rings mainly occurred due to the inhibition of extracellular Ca2+ influx through the blockade of voltage-dependent L-type Ca2+ channels and/or nonselective cation channels. These results suggest that nuciferine has a therapeutic effect on respiratory diseases associated with the aberrant contraction of airway smooth muscles and/or bronchospasm.

This study describes a universal fluorometric method for sensitive detection of analytes by using aptamers. It is based on the use of graphene oxide (GO) and cryonase-assisted signal amplification. GO is a strong quencher of FAM-labeled nucleic acid probes, while cryonase digests all types of nucleic acid probes. This makes the platform widely applicable to analytes for which the corresponding aptamers are available. Theophylline and ATP were chosen as model analytes. In the absence of targets, dye-labeled aptamers are in a flexible single strand state and adsorb on the GO. As a result, the probes are non-fluorescent due to the efficient quenching of dyes by GO. Upon the addition of a specific target, the aptamer/target complex desorbed from the GO surface and the probe becomes fluorescent. The released complex will immediately become a substrate for cryonase digestion and subsequently releasing the target to bind to another aptamer to initiate the next round of cleavage. This cyclic reaction will repeat again and again until all the related-probes are consumed and all fluorophores light up, resulting in significant fluorescent signal amplification. The detection limits are 47 nM for theophylline and 22.5 nM for ATP. This is much better than that of known methods. The assay requires only mix-and-measure steps that can be accomplished rapidly. In our perception, the detection scheme holds great promise for the design enzyme-aided amplification mechanisms for use in bioanalytical methods.

This study focused on investigating whether NS8593 reverses airway smooth muscle (ASM) contraction and the underlying mechanism.ASM contraction in mouse tracheal rings and lung slices was measured. Currents mediated by voltage dependent CaHigh KOur results indicate that NS8593 inhibits LVDCCs and NSCCs, resulting in decreases of intracellular Ca

This study describes a universal fluorometric method for sensitive detection of analytes by using aptamers. It is based on the use of graphene oxide (GO) and cryonase-assisted signal amplification. GO is a strong quencher of FAM-labeled nucleic acid probes, while cryonase digests all types of nucleic acid probes. This makes the platform widely applicable to analytes for which the corresponding aptamers are available. Theophylline and ATP were chosen as model analytes. In the absence of targets, dye-labeled aptamers are in a flexible single strand state and adsorb on the GO. As a result, the probes are non-fluorescent due to the efficient quenching of dyes by GO. Upon the addition of a specific target, the aptamer/target complex desorbed from the GO surface and the probe becomes fluorescent. The released complex will immediately become a substrate for cryonase digestion and subsequently releasing the target to bind to another aptamer to initiate the next round of cleavage. This cyclic reaction will repeat again and again until all the related-probes are consumed and all fluorophores light up, resulting in significant fluorescent signal amplification. The detection limits are 47 nM for theophylline and 22.5 nM for ATP. This is much better than that of known methods. The assay requires only mix-and-measure steps that can be accomplished rapidly. In our perception, the detection scheme holds great promise for the design enzyme-aided amplification mechanisms for use in bioanalytical methods. Graphical abstract A cryonase-assisted signal amplification (CASA) method has been developed by using graphene oxide (GO) conjugated with a fluorophore-labeled aptamer for fluorescence signal generation. It has a large scope because it may be applied to numerous analytes.

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K

Mitochondria are double-membrane-bound organelles involved mainly in supplying cellular energy, but also play roles in signaling, cell differentiation, and cell death. Mitochondria are implicated in carcinogenesis, and therefore dozens of lethal signal transduction pathways converge on these organelles. Accordingly, mitochondria provide an alternative target for cancer management. In this study, F16, a drug that targets mitochondria, and chlorambucil (CBL), which is indicated for the treatment of selected human neoplastic diseases, were covalently linked, resulting in the synthesis of a multi-mitochondrial anticancer agent, FCBL. FCBL can associate with human serum albumin (HSA) to form an HSA-FCBL nanodrug, which selectively recognizes cancer cells, but not normal cells. Systematic investigations show that FCBL partially accumulates in cancer cell mitochondria to depolarize mitochondrial membrane potential (MMP), increase reactive oxygen species (ROS), and attack mitochondrial DNA (mtDNA). With this synergistic effect on multiple mitochondrial components, the nanodrug can effectively kill cancer cells and overcome multiple drug resistance. Furthermore, based on its therapeutic window, HSA-FCBL exhibits clinically significant differential cytotoxicity between normal and malignant cells. Finally, while drug dosage and drug resistance typically limit first-line mono-chemotherapy, HSA-FCBL, with its ability to compromise mitochondrial membrane integrity and damage mtDNA, is expected to overcome those limitations to become an ideal candidate for the treatment of neoplastic disease.

The effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone, as well as the underlying mechanisms, are not clear. In this investigation, we elucidated the underlying mechanisms of the distinct effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone. In CASMCs, owing to the functional loss of Ca2+-activated Cl- (Clca) channels, Ca2+ sparks activated large-conductance Ca2+-activated K+ channels (BKs), resulting in a decreases in tone against a spontaneous depolarization-caused high tone in the resting state. In ASMCs, Ca2+ sparks induced relaxation through BKs and contraction via Clca channels. However, the integrated result was contraction because Ca2+ sparks activated BKs prior to Clca channels and Clca channels-induced depolarization was larger than BKs-caused hyperpolarization. However, the effects of Ca2+ sparks on both cell types were determined by L-type voltage-dependent Ca2+ channels (LVDCCs). In addition, compared with ASMCs, CASMCs had great and higher amplitude Ca2+ sparks, a higher density of BKs, and higher Ca2+ and voltage sensitivity of BKs. These differences enhanced the ability of Ca2+ sparks to decrease CASMC and to increase ASMC tone. The higher Ca2+ and voltage sensitivity of BKs in CASMCs than ASMCs were determined by the β1 subunits. Moreover, Ca2+ sparks showed the similar effects on human CASMC and ASMC tone. In conclusions, Ca2+ sparks decrease CASMC tone and increase ASMC tone, mediated by BKs and Clca channels, respectively, and finally determined by LVDCCs.

β2-adrenoceptor agonists are commonly used as bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), however, they induce severe side effects. Therefore, developing new bronchodilators is essential. Herbal plants were extracted and the extracts' effect on airway smooth muscle (ASM) precontraction was assessed. The ethyl alcohol extract of semen cassiae (EESC) was extracted from Semen cassia. The effects of EESC on the ACh- and 80 mM K+-induced sustained precontraction in mouse and human ASM were evaluated. Ca2+ permeant ion channel currents and intracellular Ca2+ concentration were measured. HPLC analysis was employed to determine which compound was responsible for the EESC-induced relaxation. The EESC reversibly inhibited the ACh- and 80 mM K+-induced precontraction. The sustained precontraction depends on Ca2+ influx, and it was mediated by voltage-dependent L-type Ca2+ channels (LVDCCs), store-operated channels (SOCs), TRPC3/STIM/Orai channels. These channels were inhibited by aurantio-obtusin, one component of EESC. When aurantio-obtusin removed, EESC's action disappeared. In addition, aurantio-obtusin inhibited the precontraction of mouse and human ASM and intracellular Ca2+ increases. These results indicate that Semen cassia-contained aurantio-obtusin inhibits sustained precontraction of ASM via inhibiting Ca2+-permeant ion channels, thereby, which could be used to develop new bronchodilators.

Lysine-specific demethylase 2A (KDM2A, also known as JHDM1A or FBXL11) plays an important role in regulating cell proliferation. However, the mechanisms on KDM2A controlling cell proliferation are varied among cell types, even controversial conclusions have been drawn. In order to elucidate the functions and underlying mechanisms for KDM2A controlling cell proliferation and apoptosis, we screened a KDM2A knockout HEK293T cell lines by CRISPR-Cas9 to illustrate the effects of KDM2A on both biological process. The results indicate that knocking down expression of KDM2A can significantly weaken HEK293T cell proliferation. The cell cycle analysis via flow cytometry demonstrate that knockdown expression of KDM2A will lead more cells arrested at G2/M phase. Through the RNA-seq analysis of the differential expressed genes between KDM2A knockdown HEK293T cells and wild type, we screened out that TGF-β pathway was significantly downregulated in KDM2A knockdown cells, which indicates that TGF-β signaling pathway might be the downstream target of KDM2A to regulate cell proliferation. When the KDM2A knockdown HEK293T cells were transient-transfected with KDM2A overexpression plasmid or treated by TGF-β agonist hydrochloride, the cell proliferation levels can be partial or completely rescued. However, the TGF-β inhibitor LY2109761 can significantly inhibit the KDM2A WT cells proliferation, but not the KDM2A knockdown HEK293T cells. Taken together, these findings suggested that KDM2A might be a key regulator of cell proliferation and cell cycle via impacting TGF-β signaling pathway.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca

An innovative signal amplification strategy assisted by RNase H is described here for the detection of DNA targets in a universal fashion. A tailor-made RNA molecular beacon (RMB) conjugated with a fluorescence resonance energy transfer (FRET) pair (fluorophore and quencher) was designed, characterized, and combined with the employment of RNase H. Its performance is compared to that of other nucleases including Exonuclease III and T7 exonuclease. Fluorometry, performed best at excitation/emission wavelengths of 490/520 nm, gives an amazingly low detection limit of 23 fM for target DNA. The method was verified by the determination of human hemochromatosis (HFE) gene. It is perceived that the method represents a versatile tool for the detection of a wide range of targets. Graphical Abstract An RNase H-assisted signal amplification (RASA) method for the fluorometric assay of nucleic acids has been developed by using a unique RNA molecular beacon (RNA MB) conjugated with a fluorophore (F) and quencher (Q) pair for signal generation.

Because of the serious side effects of the currently used bronchodilators, new compounds with similar functions must be developed. We screened several herbs and found that Polygonum aviculare L. contains ingredients that inhibit the precontraction of mouse and human airway smooth muscle (ASM). High K+-induced precontraction in ASM was completely inhibited by nifedipine, a selective blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). However, nifedipine only partially reduced the precontraction induced by acetylcholine chloride (ACH). Additionally, the ACH-induced precontraction was partly reduced by pyrazole-3 (Pyr3), a selective blocker of TRPC3 and stromal interaction molecule (STIM)/Orai channels. These channel-mediated currents were inhibited by the compounds present in P. aviculare extracts, suggesting that this inhibition was mediated by LVDCCs, TRPC3 and/or STIM/Orai channels. Moreover, these channel-mediated currents were inhibited by quercetin, which is present in P. aviculare extracts. Furthermore, quercetin inhibited ACH-induced precontraction in ASM. Overall, our data indicate that the ethyl acetate fraction of P. aviculare and quercetin can inhibit Ca2+-permeant LVDCCs, TRPC3 and STIM/Orai channels, which inhibits the precontraction of ASM. These findings suggest that P. aviculare could be used to develop new bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease.

Background/Aims: Bitter-tasting chloroquine can suppress T cell activation by inhibiting Ca2+ signaling. However, the mechanism of inhibition remains largely unclear. Methods: In this study, CD4+ T cells were isolated from the thymus, and the calcium content of CD4+ thymocytes was measured using fura-2 AM and a TILL imaging system. Pyrazole-3 (Pyr3), thapsigargin (TG), and caffeine were used to assess the effects of chloroquine on the intracellular Ca2+ content of CD4+ T cells. Results: In murine CD4+ thymocytes, chloroquine decreased the TG-triggered intracellular Ca2+ increase in a dose-dependent manner. In the absence of chloroquine under Ca2+-free conditions (0 mM Ca2+ and 0.5 mM EGTA), TG induced a transient Ca2+ increase. After restoration of the extracellular Ca2+ concentration to 2 mM, a dramatic Ca2+ increase occurred. This elevation was completely blocked by chloroquine and was markedly inhibited by Pyr3, a selective antagonist of transient receptor potential C3 (TRPC3) channel and stromal interaction molecule (STIM)/Orai channel. Furthermore, the TG-induced transient Ca2+ increase under Ca2+-free conditions was eliminated in the presence of chloroquine. Chloroquine also blocked the dialyzed inositol-1,4,5-trisphosphate (IP3)-induced intracellular Ca2+ increase. However, chloroquine was not able to decrease the caffeine-induced Ca2+ increase. Conclusion: These data indicate that chloroquine inhibits the elevation of intracellular Ca2+ in thymic CD4+ T cells by inhibiting IP3 receptor-mediated Ca2+ release from intracellular stores and TRPC3 channel-mediated and/or STIM/Orai channel-mediated Ca2+ influx.

Background/Aims: Tetraethylammonium chloride (TEA) induces oscillatory contractions in mouse airway smooth muscle (ASM); however, the generation and maintenance of oscillatory contractions and their role in ASM are unclear. Methods: In this study, oscillations of ASM contraction and intracellular Ca2+ were measured using force measuring and Ca2+ imaging technique, respectively. TEA, nifedipine, niflumic acid, acetylcholine chloride, lithium chloride, KB-R7943, ouabain, 2-Aminoethoxydiphenyl borate, thapsigargin, tetrodotoxin, and ryanodine were used to assess the mechanism of oscillatory contractions. Results: TEA induced depolarization, resulting in activation of L-type voltage-dependent Ca2+ channels (LVDCCs) and voltage-dependent Na+ (VNa) channels. The former mediated Ca2+ influx to trigger a contraction and the latter mediated Na+ entry to enhance the contraction via activating LVDCCs. Meanwhile, increased Ca2+-activated Cl- channels, inducing depolarization that resulted in contraction through LVDCCs. In addition, the contraction was enhanced by intracellular Ca2+ release from Ca2+ stores mediated by inositol (1,4,5)-trisphosphate receptors (IP3Rs). These pathways together produce the contractile phase of the oscillatory contractions. Furthermore, the increased Ca2+ activated the Na+-Ca2+ exchanger (NCX), which transferred Ca2+ out of and Na+ into the cells. The former induced relaxation and the latter activated Na+/K+-ATPase that induced hypopolarization to inactivate LVDCCs causing further relaxation. This can also explain the relaxant phase of the oscillatory contractions. Moreover, the depolarization induced by VNa channels and NCX might be greater than the hypopolarization caused by Na+/K+-ATPase alone, inducing LVDCC activation and resulting in further contraction. Conclusions: These data indicate that the TEA-induced oscillatory contractions were cooperatively produced by LVDCCs, VNa channels, Ca2+-activated Cl- channels, NCX, Na+/K+ ATPase, IP3Rs-mediated Ca2+ release, and extracellular Ca2+.

The effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone, as well as the underlying mechanisms, are not clear. In this investigation, we elucidated the underlying mechanisms of the distinct effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone. In CASMCs, owing to the functional loss of Ca2+-activated Cl- (Clca) channels, Ca2+ sparks activated large-conductance Ca2+-activated K+ channels (BKs), resulting in a decreases in tone against a spontaneous depolarization-caused high tone in the resting state. In ASMCs, Ca2+ sparks induced relaxation through BKs and contraction via Clca channels. However, the integrated result was contraction because Ca2+ sparks activated BKs prior to Clca channels and Clca channels-induced depolarization was larger than BKs-caused hyperpolarization. However, the effects of Ca2+ sparks on both cell types were determined by L-type voltage-dependent Ca2+ channels (LVDCCs). In addition, compared with ASMCs, CASMCs had great and higher amplitude Ca2+ sparks, a higher density of BKs, and higher Ca2+ and voltage sensitivity of BKs. These differences enhanced the ability of Ca2+ sparks to decrease CASMC and to increase ASMC tone. The higher Ca2+ and voltage sensitivity of BKs in CASMCs than ASMCs were determined by the β1 subunits. Moreover, Ca2+ sparks showed the similar effects on human CASMC and ASMC tone. In conclusions, Ca2+ sparks decrease CASMC tone and increase ASMC tone, mediated by BKs and Clca channels, respectively, and finally determined by LVDCCs.

Background Chloroquine, a bitter tastant, inhibits Ca2+ signaling, resulting in suppression of B cell activation; however, the inhibitory mechanism remains unclear. Results In this study, thapsigargin (TG), but not caffeine, induced sustained intracellular Ca2+ increases in mouse splenic primary B lymphocytes, which were markedly inhibited by chloroquine. Under Ca2+-free conditions, TG elicited transient Ca2+ increases, which additionally elevated upon the restoration of 2 mM Ca2+. The former were from release of intracellular Ca2+ store and the latter from Ca2+ influx. TG-induced release was inhibited by 2-APB (an inhibitor of inositol-3-phosphate receptors, IP3Rs) and chloroquine, and TG-caused influx was inhibited by pyrazole (Pyr3, an inhibitor of transient receptor potential C3 (TRPC3) and stromal interaction molecule (STIM)/Orai channels) and chloroquine. Moreover, chloroquine also blocked Ca2+ increases induced by the engagement of B cell receptor (BCR) with anti-IgM. Conclusions These results indicate that chloroquine inhibits Ca2+ elevations in splenic B cells through inhibiting Ca2+ permeable IP3R and TRPC3 and/or STIM/Orai channels. These findings suggest that chloroquine would be a potent immunosuppressant.

The flesh firmness of AC andrinmutant tomato fruits picked freshly were the largest. Respiration rate and ethylene production were very low at this time. With ethylene production increase, fruit firmness began to decline. 100μL/L ethephon significantly increased AC tomato fruit ethylene release, respiration rate, ACS activity and ACO activity, and decreased flesh firmness. However, there were no significant differences inrinmutant between control and ethephon treatment. It was shown RIN transcription factor regulated ethylene biosynthesis by ACC synthase and ACC oxidase.

The physicochemical properties, Ca2+–ATPase activity, and surface hydrophobicity of grass carp myobrillar with different cryoprotectants (0.5 % irradiated degraded KGM, 0.5 % β-glucanase KGM hydrolysate, 8 % sucrose-sorbitol (1:1 w/w)) were investigated. The molecular weight analysis showed that the irradiated degraded KGM and β-glucanase KGM hydrolysate were 1.209 × 104 and 2.093 × 104 Da, respectively. The solubility, Ca2+–ATPase activity, and total and reactive sulfhydryl (SH) content of treatments containing KGM hydrolysates were higher than other treatments during frozen storage. Addition of KGM hydrolysates could effectively prevent the increase of surface hydrophobicity. The gel strength and whiteness of surimi showed that the addition of KGM hydrolysates in this experiment resulted in surimi with comparable quality.

Aims This study focused on investigating whether NS8593 reverses airway smooth muscle (ASM) contraction and the underlying mechanism. Main methods ASM contraction in mouse tracheal rings and lung slices was measured. Currents mediated by voltage dependent Ca2+ channels (VDCCs) and ACH-activated channels were measured using the whole-cell patch-clamp technique in single tracheal smooth muscle cells (TSMCs). Intracellular Ca2+ level and cell length were measured using an LSM 700 laser confocal microscope and a Zen 2010 software. Mouse respiratory system resistance (Rrs) was assessed using a FlexiVent FX system. Key findings High K+ (80 mM K+) and ACH induced ASM contraction in mouse tracheal rings and lung slices, which was partially relaxed by nifedipine (blocker of L-type VDCCs, LVDCCs), YM-58483 (blocker of store-operated Ca2+ entry (SOCE), transient receptor potential C3 (TRPC3) and TRPC5 channels), respectively. However, the contraction was completely reversed by NS8593, whereas, slightly relaxed by formoterol. ACH activated inward currents, which displayed linear and reversed around 0 mV, indicating the currents were mediated by non-selective cation channels (NSCCs). Moreover, these currents were blocked by YM-58483. In addition, such currents were abolished by NS8593, implicating that NS8593 inhibits the same channels. Besides, NS8593 inhibited increases of intracellular Ca2+ and the associated cell shortening. Finally, NS8593 inhibited ACH-induced increases of mouse respirator system resistance (Rrs). Significance Our results indicate that NS8593 inhibits LVDCCs and NSCCs, resulting in decreases of intracellular Ca2+ and then leading to ASM relaxation. These data suggest that NS8593 might be a new bronchodilator.

Background Chloroquine, a bitter tastant, inhibits Ca2+ signaling, resulting in suppression of B cell activation; however, the inhibitory mechanism remains unclear. Results In this study, thapsigargin (TG), but not caffeine, induced sustained intracellular Ca2+ increases in mouse splenic primary B lymphocytes, which were markedly inhibited by chloroquine. Under Ca2+-free conditions, TG elicited transient Ca2+ increases, which additionally elevated upon the restoration of 2 mM Ca2+. The former were from release of intracellular Ca2+ store and the latter from Ca2+ influx. TG-induced release was inhibited by 2-APB (an inhibitor of inositol-3-phosphate receptors, IP3Rs) and chloroquine, and TG-caused influx was inhibited by pyrazole (Pyr3, an inhibitor of transient receptor potential C3 (TRPC3) and stromal interaction molecule (STIM)/Orai channels) and chloroquine. Moreover, chloroquine also blocked Ca2+ increases induced by the engagement of B cell receptor (BCR) with anti-IgM. Conclusions These results indicate that chloroquine inhibits Ca2+ elevations in splenic B cells through inhibiting Ca2+ permeable IP3R and TRPC3 and/or STIM/Orai channels. These findings suggest that chloroquine would be a potent immunosuppressant.

The effects of hypertonic solution on airway smooth muscle (ASM) contraction and the underlying mechanisms are largely unknown. We found that hypertonic saline (HS) inhibited acetylcholine (ACh)-induced contraction of ASM from the mouse trachea and human bronchi. In single mouse ASM cells (ASMCs), ACh induced an increase in intracellular Ca

In China, casualties and economic losses are increasing due to disasters recent year. To enhance the ability of disaster prevention and relief, it is necessary to develop the management system of disaster prevention and relief based on building information modeling. The system will be much more intuitive than before with the BIM technology and all the data can be stored in BIM model. This paper discusses about how to develop such a management system. Considering the advantage and disadvantage of B/S and C/S structure, B/S structure is used in our system. We separate the data from the model and import the data into the relational database for management. To utilize the strength of GIS, we combined the geometric solid model with triangulated irregular network model in the system. And the system also provides some useful function to the users such as disaster simulation, public education, escape route analysis etc, which can offer strong support to emergency response, command dispatching and decision analysis.

Ginger, from the rhizome of Zingiber officinale Rosco (Zingiberaceae), is a common condiment for foods and beverages. In this work, we tested a hypothesis that a steaming process affects the chemical profile and anticancer potential of ginger. An HPLC method with TOF/MS and DAD was developed to analyse the chemical constituents in ginger. The antiproliferative effect of fresh, dried and steamed gingers was evaluated using human Hela cancer cells. The results showed that the antiproliferative effect of steamed ginger at 120 °C for 4 h was approximately 1.5- and 2-fold higher than that of dried and fresh ginger, respectively. Twenty-two components were characterised in the steamed ginger. The decreased concentration of gingerols and increased levels of shogaols contributed to the improved anticancer potential of the steamed ginger. This study elucidated the relationship of the heating process with the constituents and anticancer activity, and developed an optimised processed ginger extract for chemoprevention.

Aim To study the differentially-expressed proteins for malonylastragaloside I in human leukemia U937 cells. Methods A suitable protein quantitative mode of label-free technique was established by LC Chip Q-TOF. It was found that the mode of peptide spectral intensity based on label-free quantification strategy of LC Chip Q-TOF showed high reproducibility, wide dynamic range, as well as simplicity and convenience. The optimized quantification mode of the peptide spectral intensity, normalization algorithm was applied to screen the differentially-expressed proteins for malonylastragaloside I (MA-I), a novel astragaloside from Astragalus sp., in human leukemia U937 cells. Results Fifteen differentially-expressed proteins with up or down-regulation over 2.0 folds were identified, six of which including HSC70, RPLP2, PHB2, PCNA, TCTP and ANXA11 were confirmed by Western blotting. We also observed significant activation of PARP1 and cleavage of caspases-3 and −9 by MA-I in U937 cells, and the pan-caspase inhibitor Z-VAD-FMK could reduce MA-I inhibited cell proliferation, indicating that caspase activation may be involved in MA-I induced cell toxicity. Conclusion This study has generated potentially functional proteins important for MA-I inhibited U937 cells proliferation by the optimized quantification mode of peptide spectral intensity.

Many of the ligands involved in developmental processes require HS (heparan sulfate) to modulate signal transduction. hHS6ST2 (human heparan sulfate D-glucosaminyl 6-O-sulfotransferase-2) is a Golgi-resident enzyme that usually acts on GlcA/IdoA(2S)-GlcNAc/NS disaccharide-6-sulfate modifications within the HS sequence. Emerging evidence indicates the importance of 6-O-sulfation in a number of developmental processes. However, any correlation with cancer-related events remains largely unexplored. In the present study, we found that hHS6ST2, but not other variants, was activated in human PC (pancreatic cancer). shRNA (short hairpin RNA)-mediated silencing of endogenous hHS6ST2 expression in the PC cell line PANC-1 inhibited cell invasion and migration. hHS6ST2 knockdown also resulted in markedly reduced tumorigenesis in immunocompromised mice. To specifically explore the molecular alterations resulting from depletion of hHS6ST2-generated 6-O-sulfation, we employed two-dimensional gel electrophoresis technology followed by nano-HPLC–ESI (electrospray ionization)–tandem MS to separate and identify total proteins from PC cells. Our data suggest that hHS6ST2 potentiates Notch signalling in PC cells. We also identified a role for hHS6ST2 in the growth and tumorigenicity of these cells which, at least in part, acts through Notch-mediated EMT (epithelial–mesenchymal transition) and angiogenesis. The results of the present study suggest that hHS6ST2 could be an attractive target for PC therapy.

Investigation of characteristic constituents of the roots of Glycyrrhiza uralensis Fischer led to isolation of four new triterpene glucuronides, namely uralsaponins C-F (1-4), an artificial product, namely the methyl ester of glycyrrhizin (5), as well as six known triterpene glucuronides (6-11). These new compounds were identified by 1D and 2D NMR spectroscopic analysis. The cytotoxicity of the selected compounds and their aglycones were evaluated against HeLa and MCF-7 cancer cell lines, and the preliminary structure-activity relationship was also elucidated.

Buyang Huanwu Decoction (BYHWD), is a well-known traditional Chinese preparation consisting of Radix Astragali, Radix Angelicae Sinensis, Rhizoma Ligustici Chuanxiong, Radix paeoniae Rubra, Flos Carthami, Semen Persicae and Lumbricus. An ultra-fast high-performance liquid chromatography (HPLC) method coupled with diode array detection (DAD) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) has been developed for rapid separation and structural identification of constituents in BYHWD. Using an ultra-fast HPLC system with short columns (4.6 x 50 mm, 1.8 microm), the total analysis time for this complex prescription is less than 30 min. With various fragmentor voltages in TOF/MS, accurate mass measurements (less than 5 ppm error) for molecular ions and characteristic fragment ions could represent reliable identification criteria for these compounds. Fifty-four major constituents from BYHWD sample, including four C-glycosyl quinochalcones, four flavonoid O-glycosides, sixteen isoflavones, six monoterpene glycosides, eight saponins, four organic acids and five amino acids, were identified or tentatively characterized based on their retention times, DAD and TOF/MS data. All the compounds were further assigned in the seven individual crude drugs. In conclusion, the ultra-fast HPLC with DAD-TOF/MS is a highly useful and efficient technique to separate and identify constituents in complex matrices of herbal medicines or preparations.

In this study, a novel multidimensional separation technique was developed to analyze differential plasma proteins of immunodepleted plasma in five patients with hepatocellular carcinomas (HCC), and mixed plasma of five healthy volunteers was used as control. Samples of human plasma were depleted to six most abundance proteins using an immunoaffinity chromatography Agilent MARS Spin Cartridge and then were separated under the preferred conditions by reversed phase LC. Overexpressed differential fraction of low-abundance proteins were collected and trypsined, and then the trypsined peptides were enriched and fractioned and indentified using HPLC-CHIP/MS. Reversed phase high-performance liquid chromatographic prefraction of immunodepleted human plasma coupled with HPLC-CHIP/MS was used to enhance mass spectrometry identification of low-abundance proteins, and about 27 kinds of proteins were identified. Twenty-three kinds of protein are related to various diseases or tumors, and among them, IMP3, ARNT2, and GRIP1 may prove to be potential biomarkers for the diagnosis of HCC.

To inspect the effect of main MS analysis conditions and data accuracy through matrix-assisted laser desorption ionization-time of flight-mass spectrometry and to obtain peptides mass fingerprint (PMF) for protein search results. BSA had been separated by SDS-PAGE, hydrolyzed by trypsin, and identified by MS, and PMF data were searched through MS-Fit search engine. The results showed that the sequence coverage and the peptide coverage gradually increase when laser strike numbers under 300 and numbers at 400, which appeared to descend tendency. Target protein score, amino acid sequence, and peptide coverage appeared to increase following increase in laser intensity, and when laser intensity was at 1413, there was a tendency to descend. Peptide coverage showed an increasing tendency following delay-time increase, and when delay time was at 260 ns, there was a tendency to descend. On the basis of optimized analysis conditions and on checking the same site for 3 times, the result showed that reproducibility was better. To inspect the accuracy of MS data, Mix 2 was used as external standard to remedy MS primitive data. It was found to score higher, order ahead, detect fewer numbers, and show better reliability as the MS data approached standard value. In contrast, we almost didn't get target protein. The results showed that MS accuracy and analysis conditions were present, which distinguished the influence on protein database search.

Flavonoids, one of the largest groups of secondary metabolites, are widespread in vegetable crops such as herbs, fruits, vegetables, grains, seeds and derived foods such as juices, wines, oils, etc. They receive considerable attention due to their biological and physiological importance. Hundreds of publications on the analysis of flavonoids have appeared over the past decade. Traditional and more advanced techniques have come to prominence for sample preparation, separation, detection, and identification. This review intends to provide an updated, concise overview on the recent development and trends of separation, identification and quantification for flavonoids by modern chromatographic and spectrophotometric analytical techniques, including gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). The sample preparation before analysis is also briefly summarized.